Bupropion (BUP) is widely used for the treatment of major depression and seasonal affective disorder and as a smoking cessation aid. Marked variability in clinical response among patients and clinically important CYP2D6- dependent drug-drug interactions (DDIs) often compromise effective and safe BUP use. Our long-term goal is to improve BUP therapy through identification of mechanisms influencing BUP's efficacy, toxicity and DDIs. As a first step, we will comprehensively elucidate the sequential stereoselective metabolism and disposition of BUP and identify mechanisms underlying CYP2D6-dependent DDIs. BUP biotransformation to 4-hydroxyBUP and threo-/erythro-hydroBUP by CYP2B6 and carbonyl reductases, respectively, is a major determinant of BUP's clinical efficacy, toxicity and DDIs. These metabolites exhibit considerable accumulation in plasma compared to BUP with widely varying plasma exposures among patients and this variability predicts treatment outcomes and side effects. To date, specific mechanisms responsible for this inter-patient variability remain unknown. BUP is administered as a racemic mixture. Its biotransformation to the active metabolites introduces additional chiral centers, generating multiple diastereomers with unique pharmacological profiles. Insights into the complex metabolism of BUP would be a major advance to the understanding of variable active metabolites disposition, effect and DDIs. A critical barrier to evaluating stereoselective metabolism of BUP has been the lack of reliable analytical methods that allow baseline resolution and quantification of different stereoisomers of the primary and secondary metabolites of BUP. As detailed in the Preliminary Studies section, we overcame this critical barrier by developing and validating the first novel chiral and achiral LC-MS/MS methods and demonstrated marked stereoselective metabolism and disposition in vitro and in vivo. In this proposal, mechanisms underlying the stereoselective sequential metabolism and disposition of BUP and associated CYP2D6-dependent DDIs will be identified. In Aim 1, critical pathways and enzymes involved in the stereoselective BUP clearance leading to formation and subsequent elimination of its active metabolites will be identified using human hepatic and extrahepatic sub-cellular fractions. In Aim 2, the inhibitory potency of stereoisomers of BUP and metabolites towards CYP2D6 activity will be tested in human liver microsomes and expressed CYP2D6. A comprehensive stepwise PBPK modeling approach will be utilized in Aim 3 to quantitatively describe steady state BUP and metabolite disposition and predict in vivo BUP DDIs. In Aim 4, the steady-state stereoselective disposition of BUP and metabolites and the time course (onset), extent (maximum inhibition) and offset of CYP2D6 inhibition will be tested in healthy volunteers. Insights gained from these integrated in vitro, in silico and in vivo studies on the complex stereospecific sequential metabolism and disposition and on the mechanisms of DDIs will advance our understanding of bupropion's efficacy, toxicity and CYP2D6-dependent DDIs, and eventually improve bupropion therapy and manage adverse DDIs.